Jacquelynn E. Larson

Cloning, characterization, and properties of seven triplet repeat DNA sequences.

Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG·CAG, CGG·CCG, or AAG·CTT) in or near the responsible genes. We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA·TAC, GAT·ATC, GTT·AAC, CAC·GTG, AGG·CCT, TCG·CGA, and AAG·CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D. (1996) J. Biol. Chem. 271, 16784-16791) describes studies on TTA·TAA. This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of ∼16 up to 158 triplets in both orientations with varying extents of polymorphisms. The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations. AAG·CTT and AGG·CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non-B structures. However, long tracts of TCG·CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG·CAG and CGG·CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R. D. (1995) J. Biol. Chem. 270, 27014-27021). This work along with other studies (Wells, R. D. (1996) J. Biol. Chem. 271, 2875-2878) on CTG·CAG, CGG·CCG, and TTA·TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations. A model to explain the reduction in mRNA abundance in Friedreichs ataxia based on intermolecular triplex formation is proposed.

FLEXIBLE DNA : GENETICALLY UNSTABLE CTG.CAG AND CGG.CCG FROM HUMAN HEREDITARY NEUROMUSCULAR DISEASE GENES

The properties of duplex CTG·CAG and CGG·CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 × 10−19 erg·cm for CTG and 1.27 × 10−19 erg·cm for CGG, ∼40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were ∼60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2.3 × 10−19 erg·cm and 10.4 base pairs (bp)/turn for CTG and 2.4 × 10−19 erg·cm and 10.3 bp/turn for CGG, respectively, all within the range for random B-DNA. Determination of the apparent helical repeat by the band shift assay indicated that the writhe of the repeats was different from that of random B-DNA. In addition, molecules of 224–245 bp in length (64–71 triplet repeats) were able to form topological isomers upon cyclization. The low bending moduli are consistent with predictions from crystallographic variations in slide, roll, and tilt. No unpaired bases or non-B-DNA structures could be detected by chemical and enzymatic probe analyses, two-dimensional agarose gel electrophoresis, and immunological studies. Hence, CTG and CGG are more flexible and highly writhed than random B-DNA and thus would be expected to act as sinks for the accumulation of superhelical density.

Abundance and length of simple repeats in vertebrate genomes are determined by their structural properties

Microsatellites are abundant in vertebrate genomes, but their sequence representation and length distributions vary greatly within each family of repeats (e.g., tetranucleotides). Biophysical studies of 82 synthetic single-stranded oligonucleotides comprising all tetra- and trinucleotide repeats revealed an inverse correlation between the stability of folded-back hairpin and quadruplex structures and the sequence representation for repeats > or =30 bp in length in nine vertebrate genomes. Alternatively, the predicted energies of base-stacking interactions correlated directly with the longest length distributions in vertebrate genomes. Genome-wide analyses indicated that unstable sequences, such as CAG:CTG and CCG:CGG, were over-represented in coding regions and that micro/minisatellites were recruited in genes involved in transcription and signaling pathways, particularly in the nervous system. Microsatellite instability (MSI) is a hallmark of cancer, and length polymorphism within genes can confer susceptibility to inherited disease. Sequences that manifest the highest MSI values also displayed the strongest base-stacking interactions; analyses of 62 tri- and tetranucleotide repeat-containing genes associated with human genetic disease revealed enrichments similar to those noted for micro/minisatellite-containing genes. We conclude that DNA structure and base-stacking determined the number and length distributions of microsatellite repeats in vertebrate genomes over evolutionary time and that micro/minisatellites have been recruited to participate in both gene and protein function.

Recombinant human DNA (cytosine-5) methyltransferase. III. Allosteric control, reaction order, and influence of plasmid topology and triplet repeat length on methylation of the fragile X CGG.CCG sequence.

Steady-state kinetic analyses revealed that the methylation reaction of the human DNA (cytosine-5) methyltransferase 1 (DNMT1) is repressed by the N-terminal domain comprising the first 501 amino acids, and that repression is relieved when methylated DNA binds to this region. DNMT1 lacking the first 501 amino acids retains its preference for hemimethylated DNA. The methylation reaction proceeds by a sequential mechanism, and either substrate (S-adenosyl-l-methionine and unmethylated DNA) may be the first to bind to the active site. However, initial binding of S-adenosyl-l-methionine is preferred. The binding affinities of DNA for both the regulatory and the catalytic sites increase in the presence of methylated CpG dinucleotides and vary considerably (more than one hundred times) according to DNA sequence. DNA topology strongly influences the reaction rates, which increased with increasing negative superhelical tension. These kinetic data are consistent with the role of DNMT1 in maintaining the methylation patterns throughout development and suggest that the enzyme may be involved in the etiology of fragile X, a syndrome characterized byde novo methylation of a greatly expanded CGG·CCG triplet repeat sequence.

An Intramolecular Triplex in the Human γ-Globin 5′-Flanking Region Is Altered by Point Mutations Associated with Hereditary Persistence of Fetal Hemoglobin

The properties of an intramolecular triplex formed in vitro at the 5′-flanking region of the human γ-globin genes were studied by chemical and physical probes. Chemical modifications performed with osmium tetroxide, chloroacetaldehyde, and diethyl pyrocarbonate revealed the presence of non-paired nucleotides on the “coding strand” at positions −209 through −217. These reactivities were induced by negative supercoiling, low pH, and magnesium ions. Downstream point mutations associated with hereditary persistence of fetal hemoglobin (HPFH) altered the extent of the modifications and some of the patterns. Specifically, C−202 → G and C−202 → T significantly decreased the reactivities, whereas the patterns were increased and altered in the T−198 → C. C−196 → T and C−195 → G caused local decreases in reactivity. Modifications at the upstream flanking duplex were modulated by the composition of the vector sequence. In summary, our data indicates the formation of an intramolecular triplex between nucleotides −209 to −217 of the “non-coding strand” and the downstream sequence containing the HPFH mutations. All of the HPFH point mutations altered the structure. More than one sequence alignment is possible for each of the triplexes. In addition, a consequence of some of the point mutations may be to facilitate slippage of the third strand relative to the Watson-Crick duplex.

TTA·TAA Triplet Repeats in Plasmids Form a Non-H Bonded Structure

CTG·CAG, CGG·CCG, and AAG·CTT triplet repeats proximal to or in disease genes expand by a non-Mendelian genetic process to cause several human hereditary syndromes. As part of our physical, biological, and genetic studies on the 10 possible triplet repeats, we discovered that the TTA·TAA repeat, isolated from the upstream region of the variant surface glycoprotein gene of Trypanosoma brucei, shows a propensity to adopt a non-H bonded structure under appropriate conditions. The other nine triplet repeat sequences do not exhibit this property. (TTA·TAA)n, where n = 90, 60, 30, and 18, cloned into pUC19 was studied by chemical and enzymatic probes as well as two-dimensional gel electrophoretic analyses under a variety of conditions. The helix opening was observed for all four inserts in supercoiled plasmids as a function of temperature, pH, metal ions, and buffer conditions using OsO4, diethyl pyrocarbonate, and chloroacetaldehyde probes. This unusual property of the TTA·TAA repeat suggests that it plays a different role from the other nine triplet repeats in gene expression.

The Myotonic Dystrophy Type 1 Triplet Repeat Sequence Induces Gross Deletions and Inversions

The capacity of (CTG·CAG)n and (GAA·TTC)n repeat tracts in plasmids to induce mutations in DNA flanking regions was evaluated in Escherichia coli. Long repeats of these sequences are involved in the etiology of myotonic dystrophy type 1 and Friedreichs ataxia, respectively. Long (CTG·CAG)n (where n = 98 and 175) caused the deletion of most, or all, of the repeats and the flanking GFP gene. Deletions of 0.6–1.8 kbp were found as well as inversions. Shorter repeat tracts (where n = 0 or 17) were essentially inert, as observed for the (GAA·TTC)176-containing plasmid. The orientation of the triplet repeat sequence (TRS) relative to the unidirectional origin of replication had a pronounced effect, signaling the participation of replication and/or repair systems. Also, when the TRS was transcribed, the level of deletions was greatly elevated. Under certain conditions, 30–50% of the products contained gross deletions. DNA sequence analyses of the breakpoint junctions in 47 deletions revealed the presence of 1–8-bp direct or inverted homologies in all cases. Also, the presence of non-B folded conformations (i.e. slipped structures, cruciforms, or triplexes) at or near the breakpoints was predicted in all cases. This genetic behavior, which was previously unrecognized for a TRS, may provide the basis for a new type of instability of the myotonic dystrophy protein kinase (DMPK) gene in patients with a full mutation.

Non-B DNA Conformations Formed by Long Repeating Tracts of Myotonic Dystrophy Type 1, Myotonic Dystrophy Type 2, and Friedreich's Ataxia Genes, Not the Sequences per se, Promote Mutagenesis in Flanking Regions

The expansions of long repeating tracts of CTG·CAG, CCTG·CAGG, and GAA·TTC are integral to the etiology of myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), and Friedreichs ataxia (FRDA). Essentially all studies on the molecular mechanisms of this expansion process invoke an important role for non-B DNA conformations which may be adopted by these repeat sequences. We have directly evaluated the role(s) of the repeating sequences per se, or of the non-B DNA conformations formed by these sequences, in the mutagenic process. Studies in Escherichia coli and three types of mammalian (COS-7, CV-1, and HEK-293) fibroblast-like cells revealed that conditions which promoted the formation of the non-B DNA structures enhanced the genetic instabilities, both within the repeat sequences and in the flanking sequences of up to ∼4 kbp. The three strategies utilized included: the in vivo modulation of global negative supercoil density using topA and gyrB mutant E. coli strains; the in vivo cleavage of hairpin loops, which are an obligate consequence of slipped-strand structures, cruciforms, and intramolecular triplexes, by inactivation of the SbcC protein; and by genetic instability studies with plasmids containing long repeating sequence inserts that do, and do not, adopt non-B DNA structures in vitro. Hence, non-B DNA conformations are critical for these mutagenesis mechanisms.

Fractionation of dG homooligonucleotides using RPC-5 column chromatography at high pH

Abstract dG oligomers, prepared by partial acid hydrolysis of dG n , were fractionated in large quantity according to chain length by RPC-5 column chromatography at high pH (0.1 m NaOH). Resolution of oligonucleotides up to approximately the 30-mer was achieved. The oligomers were characterized by partial chemical digestion and analysis on 20% polyacrylamide-urea gels.

CHAPTER 46 – Gross Rearrangements Caused by Long Triplet and Other Repeat Sequences

This chapter focuses on gross rearrangements caused by long triplet and other repeat sequences. The most fascinating and unique feature of TRSs and other repeat sequences in DNA is their ability to adopt alternative conformations that differ dramatically from the commonly known, right-handed, antiparallel, double helix, generally referred to as B-DNA. The formation of non-B conformations in vivo is mostly based on the behavior of the DNA sequences in vitro, their relationships with DNA topology, and in certain cases antibody binding. Some of most relevant nonB-DNA are slipped (hairpin) structures, cruciforms, triplexes, tetraplexes and/-motifs, and left-handed Z-DNA are formed in chromosomes and elicit profound genetic consequences via recombination repair. On the other hand, repeating sequences, probably in their nonB conformations, cause gross genomic rearrangements such as deletions, insertions, inversions, translocations, and duplications. These rearrangements are the genetic basis for scores of human diseases, including polycystic kidney disease, adrenoleukodystrophy, follicular lymphomas, and spermatogenic failure.